Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)

Abstract

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.